1. Technical Field
The present invention relates to a device and a method for dielectrophoretic manipulation of suspended particulate matter. In addition the invention relates to a method for production of the device.
2. Background Information
Within the context of the present application, the word “comprises” is taken to mean “includes among other things,” and is not taken to mean “consists of only.”
The terms electrically “non-conductive” and “insulating” as used herein are interchangeable and have the same meaning. They are interpreted to mean “substantially electrically non-conductive.”
The term “manipulation” is interpreted to include known laboratory or plant techniques including analysis, filtration, fractionation, collection or separation.
Dielectrophoresis (DEP) is a well known technique for separation based on the manipulation of particles in non-uniform electric fields. It can be used for separation of particles, either by binary separation of particles into two separate groups, or for fractionation of many populations. It can also be used for the collection of particles and for transport of particles along an electrode array. It is based generally on exploitation of differences in the dielectric properties of populations of particles. This enables a heterogeneous mix of particles to be fractionated by exploiting small differences in polarizability or by using a dielectrophoretic force in conjunction with other factors such as imposed flow or particle diffusion.
If a dielectric particle is suspended in an electric field, it will polarize and there is an induced dipole. The magnitude and direction of this induced dipole depends on the frequency and magnitude of the applied electric field, and the dielectric properties of particle and medium. The interaction between the induced dipole and the electric field can generate movement of the particle, the nature of which depends on a number of factors including the extent to which the field is non-uniform both in terms of magnitude and phase.
If the electric field is uniform, the attraction between the dipolar charges and the electric field is equal and opposite and the result is no net movement, unless the particle carries a net charge and the field frequency is equal to, or near, zero. However, if the field is spatially non-uniform, the magnitude of the forces on either side of the particle will be different, and a net force exists in the direction in which the field magnitude is greatest. Since the direction of force is governed by the spatial variation in field strength, the particle will always move along the direction in which the electric field increases by the greatest amount; that is, it moves along the direction of greatest increasing electric field gradient regardless of field polarity. Since the direction of motion is independent of the direction of the electric field polarity, it is observed for both AC and DC fields; the dipole re-orients with the applied field polarity, and the force is always governed by the field gradient rather than the field orientation. The magnitude and direction of the force along this vector is a complex function of the dielectric properties of particle and medium. If a force exists in a direction of increasing field gradient, it is termed positive DEP. Its opposite effect, negative DEP, acts to repel a particle from regions of high electric field, moving it “down” the field gradient. Whether a particle experiences positive or negative DEP is dependent on its polarizability relative to its surrounding medium; differences in the quantity of induced charge at the interface between particle and medium lead to dipoles oriented counter to the applied field (and hence positive DEP) where the polarizability of a particle is more than that of the medium, and in the same direction as an applied field (and hence negative DEP) where it is less. Since relative polarizability is a complex function dependent not only on the permitivity and conductivity of the particle and medium, but also on the applied field frequency, it has a strong frequency dependence and particles may experience different dielectrophoretic behavior at different frequencies.
Where there are non-uniformities in phase, a different but related phenomenon is observed. An electric field having a peak which moves through space over a time can be described as a wave whose phase varies with position. Where an electric field moves across the particle, a dipole is induced that also moves. If the velocity of the field across a particle is sufficiently high, then the dipole (which takes a finite time to respond to the field, dictated by its dielectric relaxation time) will lag behind it at a finite distance; the interaction between peaks in an electric field and the physically displaced dipole induces a force which acts on the particle. The direction of the force is dependent on polarizability: if the particle is more polarizable than the medium then the dipole aligns counter to the electric field, causing an attractive force to be induced resulting in the particle moving in the same direction of movement as the local applied field; if the particle is less polarizable than the medium then the dipole (and net particle motion) are reversed. Similarly, if the displacement of the dipole is greater than half the wavelength of the electric field as it moves through space, then it will interact with a preceding field maximum resulting in a reversal of direction. The name given to this effect is traveling wave dielectrophoresis (TWD). Since it is possible to generate an electric field with spatially variant electric field magnitude and phase, a particle suspended in such a field will experience both DEP and TWD simultaneously, with the vectors of force acting (i) along the direction of a maximum change in electric field; and (ii) along the direction of a maximum change in field phase.
DEP can be used for detection, fractionation, concentration or separation of complex particles. Additionally, studying the DEP behavior of particles at different frequencies can allow the study of the dielectric properties of those particles. For example, it can be used to examine changes in cell cytoplasm in cells after infection by a virus. This potentially enables detection where the differences between cell types are subtle and could be applied to the separation or detection of cancerous or healthy cells, viable or non-viable cells, leukaemic cells in blood, different species of bacteria and placental cells from maternal blood.
Thus, it is clear that DEP can be a versatile technique for detection, analysis, fractionation, concentration or separation. In view of this, significant interest is being invested in dielectrophoresis technology. However, at present DEP is based on planar two dimensional technology, developed for the silicon chip industry. The known electrodes (usually gold) are fabricated from thin layer films (typically up to 1 μm thick) on a glass substrate (e.g., a microscope slide). They are expensive to produce, and the volume above the electrodes in which the electric field penetrates is limited to a few tens of microns, meaning the overall volume of sample is small and the effectiveness of the known devices is severely limited. Thus, there is a need for a new device for dielectrophoretic separation of suspended particulate matter.
High throughput screening is conventionally used to evaluate a large number of candidate compounds for their possible use as pharmaceutical drugs. To do this, experiments are often carried out on living cells (e.g., bacteria or tissue cultures), which are subjected to small amounts of possible candidate chemicals and monitored to check for desired changes. Monitoring is carried out using several known techniques, e.g., selective chemical staining or monitoring pH changes with chemical indicators. To perform a large number of experiments in the quickest possible time they are carried out in parallel and to save on reagents the experiments are generally carried out in well plates. These plates have a large number of small wells wherein each well can be used to contain the reagents for performing one experiment. Known plates have 384 or 1536 wells, while each well is capable of containing only a few microliters of sample. To perform even more parallel experiments with even smaller samples new plates having even more wells are currently under development.
Finding a technique for assessing the results of experiments performed in such a small volume can be difficult, especially since most known detection methods require the presence of an indicator or dye that might itself interact with the organism or the drug candidate. Therefore, DEP can be a valuable tool to evaluate these assays since it can detect changes in the morphology of cells without any marker chemicals. In view of the fact that DEP can separate particles based on their dielectric properties, bacteria or cells can be detected based on properties of the cell wall or membrane. This can be used for bioassays to evaluate whether a drug candidate interacts with a receptor at the cell wall or membrane. However, because conventional DEP assays are performed with flat two dimensional electrode structures the electric field generated by the electrodes does not penetrate sufficiently far into liquid media and therefore until now it has only been possible to probe a very small sample volume. Therefore, there is a need for a new electrode structure that can be used to probe a larger volume within a small well to allow quick analysis of a sample of several micro-liters.